title: Agaritine from Agaricus blazei Murrill induces apoptosis in the leukemic cell line U937.
abstract: BACKGROUND
  Agaricus blazei Murrill (ABM) has been shown to exhibit immunostimulatory and anti-cancer activities; however, its mechanism of action is poorly understood. We recently found that the diffusible fraction of hot-water extract of ABM exhibits anti-tumor activity toward leukemic cells, and identified it as agaritine, a hydrazine-containing compound. In the present study, we examined the morphological and cytochemical effects of agaritine on U937 cells to elucidate the tumoricidal mechanism of agaritine.
  
  
  METHODS
  Surface expression of phosphatidylserine (evaluated by annexin V binding), Fas antigen, DNA cleavage using TUNEL staining, changes in caspase activities and cytochrome c release, before and after treatment with agaritine, were examined using U937 cells.
  
  
  RESULTS
  Nuclear damage, DNA fragmentation, was observed by Wright-Giemsa, TUNEL staining and agarose gel electrophoresis when U937 cells were incubated with 10μg/mL of agaritine for 48h. Flow cytometric analysis indicated that agaritine augments the proportion of annexin V-positive U937 cells without significant change in Fas antigen expression. Activities of caspase-3, -8 and -9 were gradually increased after the addition of agaritine. In the presence of caspase-3 or granzyme B inhibitor, except for the caspase-8 inhibitor, annexin V expression was significantly decreased, suggesting that mainly caspase-3 and -9 participate in the apoptotic pathway. Furthermore, cytochrome c release was detected by western blotting analysis after agaritine treatment.
  
  
  CONCLUSIONS
  These results strongly suggest that the ABM constituent agaritine moderately induces apoptosis in U937 leukemic cells via caspase activation through cytochrome c release from mitochondria.
  
  
  CONCLUSIONS
  This is the first report suggesting that the anti-tumor effect of agaritine is mediated through apoptosis. The present results might provide helpful suggestions for the design of anti-tumor drugs toward leukemia patients.
authors.affiliation: Department of Clinical Hematology, Fujita Health University School of Health Sciences, Toyoake, Aichi 470-1192, Japan. hakiyama@fujita-hu.ac.jp
authors.forename: Hidehiko
authors.initials: H
authors.lastname: Akiyama
authors.forename: Masahiro
authors.initials: M
authors.lastname: Endo
authors.forename: Taei
authors.initials: T
authors.lastname: Matsui
authors.forename: Itsurou
authors.initials: I
authors.lastname: Katsuda
authors.forename: Nobuhiko
authors.initials: N
authors.lastname: Emi
authors.forename: Yasuko
authors.initials: Y
authors.lastname: Kawamoto
authors.forename: Takaaki
authors.initials: T
authors.lastname: Koike
authors.forename: Hidehiko
authors.initials: H
authors.lastname: Beppu
mesh_terms: D000364:Agaricus / Q000737:chemistry*; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism; D053181:Caspase 8 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D045304:Cytochromes c / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects; D005434:Flow Cytometry; D006801:Humans; D020287:In Situ Nick-End Labeling; D016403:Lymphoma, Large B-Cell, Diffuse / Q000378:metabolism / Q000473:pathology; D015394:Molecular Structure; D010659:Phenylhydrazines / Q000737:chemistry / Q000494:pharmacology*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors; D020298:U937 Cells; D019014:fas Receptor / Q000378:metabolism
chemical_list: D010659:Phenylhydrazines; D019014:fas Receptor; D045304:Cytochromes c; D011065:Poly(ADP-ribose) Polymerases; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; C015654:agaritine